Professor of Biochemistry Dana Carroll, Ph.D.,
one of the originators of Zinc Finger Nuclease
technology for genome editing at the University of Utah

CompoZr® Zinc Finger Nucleases (ZFNs) are the newest, most efficient, method for genome editing, and are now available from Sigma® Life Science. One of the researchers who made this technology possible is Dr. Dana Carroll of the University of Utah. His lab demonstrated that ZFNs have the capability to make targeted double-strand breaks on chromosomes within living cells.

Strangely enough, Dr. Carroll's early science studies were focused on chemistry, physics and mathematics. It was not until he started graduate school that he began to attend seminars in biology.

Dana Carroll:

(At first) I didn't know the vocabulary. After a while I learned to speak the language, and I decided I was more interested in biology and the applications of biochemistry than I was in straight chemistry.

From the time Dr. Carroll started in biology, site-specific DNA breaks were of high interest to him. In the early to mid-1990s, his lab was working with triplex forming oligos, which bind in the major groove of duplex DNA with high specificity and affinity. Therefore, when the lab of Dr. Srinivasan Chandrasegaran demonstrated that site-specific DNA cleavage could be achieved by attaching FokI to novel DNA binding proteins, Dana saw the potential for collaboration.

Dana Carroll:

In the early 1990s, we were already trying to develop reagents that could make site specific breaks in DNA. We knew that double-strand breaks could be recombinogenic, and so we were playing around with triplex forming oligos. When Dr. Chandrasegaran's paper appeared in 1996, we were already primed to understand the potential of these hybrid enzymes. I called Chandra right after his paper came out. I had never met him before, but I called him and suggested we collaborate to study whether these hybrid proteins could stimulate recombination. And that's how we got started. We already knew about double-strand breaks in DNA stimulating recombination, and we knew there was potential to manipulate the DNA binding modules of zinc fingers. So, when that paper came out, we were ready.

Dr. Carroll is quick to give credit to those who have come before him in the field, making his accomplishments possible. We asked him who some of his inspirations were. The list is long and impressive, and shows how several scientists have contributed to illuminating the complexities of biology.

"Technologies which are now commonplace in laboratories across the world had to be discovered, then optimized for all to use. This is true for DNA isolation and purification, PCR, etc." – Dr. Carroll

Dana Carroll:

I have a huge amount of admiration for a lot of people in science. One is Don Brown, who I did a postdoc with back in the early 1970s. He was somebody who worked in the lab throughout his career, and was a very serious scientist, not interested in promoting himself. He was the one who characterized the first eukaryotic transcription factor, TF3A, and this is actually where zinc fingers were first identified. Aaron Klug's group found that these repetitive modules bind zinc and are involved in DNA recognition.

And another person (I admire) is Joe Gall. He's the same generation as Don Brown, and if you look back over his career, he developed in situ hybridization and was the first to isolate and sequence telomeres. He knows so much about biology that, when he has a particular problem, he can choose the organism that he thinks is best suited to solving that problem.

Not able to stop at just two people, Dr. Carroll added Mario Capecchi, 2007 Nobel Prize winner in Physiology or Medicine for developing gene targeting in mouse ES cells.

Dana Carroll:

I have a huge amount of admiration for Mario. He doesn't let himself get distracted by irrelevant things. He went after the problem of doing recombination in mammalian cells, despite people thinking that they would never work. He developed the technique that people are using all over the world now.

In talking with Dr. Carroll, and looking at his website (www.biochem.utah.edu/carroll/), it's clear that collaborations play a large part in his research. We asked him to comment on the role that collaboration plays in his work.

Dana Carroll:

Collaboration is a way to enhance a single lab’s capabilities. In my recent experience, working with others has allowed us to try the ZFN gene targeting technology in quite a number of different organisms. My scientific outlook has been broadened enormously, as I learn about the particular features of these various systems. In some cases, these collaborations have led to publications with people who were already friends, and in others they have led to the development of valuable and enjoyable new friendships. I am particularly grateful to the many people who have been willing to share their knowledge, and to devote their time to projects I find interesting.

The discovery that ZFNs can be used to make such specific mutations has a huge impact for the overall genetics research community. We asked Dr. Carroll how ZFNs are affecting the world of science now, and implications for the future.

Dana Carroll:

People can now contemplate making very specific mutations in their genes of interest, which was only previously possible in yeast and some simple organisms, and in mice. With ZFN technology, this targeting capability is available for lots of different organisms. So that's been a big change for geneticists, but the field is still expanding. The number of applications is still increasing as we learn more about how we can use them. And so it’s still in the growth phase, which is very exciting.

As we discussed the possible applications for zinc fingers, Dr. Carroll told us that he talks to ZFN users across the world, and commented on some of the work that has been done in the different organisms.

“People can now contemplate making very specific mutations in their genes of interest, which was only previously possible in yeast and some simple organisms, and in mice.” – Dr. Carroll

Dana Carroll:

People are starting to work on larger agricultural animals like pigs and cows, to study disease and to make genetic modifications. For example, pigs are used as a source of organs for human transplantation, and work is now being done to engineer transplant organs to make them more compatible with humans. These organs have cell surface products that are immunogenic in humans, and so (the) knockout of these markers could reduce the frequency of organ rejection.

Gene therapy is another area where ZFNs are undergoing initial testing. Right now, the only practical way to use the ZFNs for human therapy is to treat cells outside the body, then put them back. Sangamo Biosciences Inc. has a clinical trial in the wings for treatment of T-cell precursors that can be reintroduced to the body after they've had the CCR5 gene knocked out, making them resistant to infection by HIV. Until such clinical trials have really been done extensively, we just don’t know what the unexpected effects really are. I’m sure that the people at Sangamo are holding their breath.

Sigma® Life Science acquired a license from Sangamo Biosciences to develop and distribute ZFNs in 2007. We had to ask... what does Dr. Carroll feel is Sigma's role in the use of the ZFN technology?

Dana Carroll:

I recently spoke at a Sigma Life Science-sponsored Targeted Genome Editing workshop, and I was impressed with a few things, including the number of people at the workshop, and the way that Sigma gave people the chance to try things out in the lab, doing real experiments.

Sigma's commitment to ZFN technology has also amazed me; the quality of the scientists who were working on it, and the way they have adapted the Sangamo technology by keeping in constant contact and finding the best way to make zinc finger sets. I was also impressed with their ambition to stay ahead of the curve... making cell lines and animals that people will want. By the time the ability to make a knockout rat using ZFNs had been published, Sigma had already created a facility to create rats for people who want them.

Until Sigma got into the game, the only way to get access to Sangamo's databases and assays was to collaborate with them. As a result, they were overwhelmed with requests, and had to be selective in whom they collaborate with. Sigma Life Science making ZFNs available to everybody really is a great contribution to the field.